Cannabinol-Mediated Inhibition of Nuclear Factor-kB, cAMP Response Element-Binding Protein, and Interleukin-2 Secretion by Activated Thymocytes

Cannabinol (CBN), an immunosuppressive cannabinoid and ligand for the peripheral cannabinoid receptor CB2, inhibits the cAMP signaling cascade in forskolin-stimulated thymocytes. The objective of the present studies was to further characterize the mechanism of CBN immune modulation by investigating its effects on interleukin-2 (IL-2) secretion, cAMP response element (CRE), and kB DNA binding activity in phorbol ester (phorbol-12-myristate-13-acetate, PMA) plus calcium ionophore (PMA/Io)-activated thymocytes. PMA/Io treatment induced CRE and kB DNA binding activity that was attenuated in the presence of CBN. A concomitant and concentration-related inhibition of IL-2 also was produced by CBN in PMA/Io-activated thymocytes. PMA/Io induced two CRE DNA binding complexes, a major complex consisting of a cAMP response element-binding protein (CREB)-1 homodimer, and a minor CREB-1/activating transcription factor (ATF)-2 complex. Both CRE complexes were inhibited by CBN. Conversely, two kB DNA binding complexes were observed, but only one was PMA/Io-inducible. However, the DNA binding activity of both complexes was diminished in the presence of CBN. The PMA/ Io-inducible kB complex was a p65/c-Rel heterodimer. Analysis of up-stream regulation revealed a decrease in phosphorylated CREB/ATF nuclear proteins in PMA/Io-activated thymocytes after CBN treatment. Similarly, CBN prevented the phosphorylation-dependent degradation of the nuclear factor-kB inhibitory protein IkB-a. These results provide a potential link between the CBN-mediated inhibition of thymocyte function, including IL-2 production, and the inhibition of two critical transcription factor families, CREB/ATF and NF-kB/Rel. Immune suppression by cannabinoid compounds is thought to be mediated, at least in part, through cannabinoid receptors (CB) expressed by leukocytes. CB1 and CB2, the two major types of cannabinoid receptors, belong to the G protein-coupled receptor superfamily and negatively regulate adenylate cyclase. CB1 is the predominant cannabinoid receptor expressed in the brain (Matsuda et al., 1990), whereas CB2 is primarily expressed by cells of the immune system (Munro et al., 1993; Schatz et al., 1997). Ligand binding to either CB1 or CB2 inhibits adenylate cyclase, thereby decreasing intracellular cAMP (Howlett et al., 1986; Kaminski et al., 1994). Cannabinol (CBN), a cannabinoid that possesses minimal central nervous system activity, exhibits higher binding affinity for the CB2 receptor (Munro et al., 1993; Schatz et al., 1997) and has recently been reported to inhibit T cell and B cell proliferation and IgM antibody responses in a dose-dependent manner (Herring et al., 1998). At comparable concentrations, CBN also decreased intracellular cAMP, protein kinase A (PKA) activity, and protein binding to a cAMP response element (CRE) after forskolin stimulation (Herring et al., 1998). Increases in intracellular cAMP facilitate the release of the catalytic subunits of PKA. The cAMP response element-binding protein (CREB)/activating transcription factor (ATF) family of transcription factors is a critical nuclear target of PKA-mediated phosphorylation and is composed of several proteins, including CREB-1, CREB-2, ATF-1, ATF-2, and CRE modulator. These transcription factors can form homodimers or heterodimers and bind to CRE motifs in the promoter region of cAMP-responsive genes. Recent studies have found interleukin-2 (IL-2) to be sensitive to inhibition by cannabinoids (Condie et al., 1996). IL-2 Received for publication February 16, 1999. 1 This work was supported by National Institute on Drug Abuse Grant DA07908. ABBREVIATIONS: CB, cannabinoid receptor; CBN, cannabinol; PKA, protein kinase A; CRE, cAMP response element; CREB, cAMP response element-binding protein; ATF, activating transcription factor; IL-2, interleukin-2; NF, nuclear factor; AT, activated T cells; AP-1, activator protein-1; PMA, phorbol-12-myristate-13 acetate; THC, tetrahydrocannabinol; EMSA, electrophoretic mobility shift assay; ELISA, enzyme-linked immunosorbent assay; MAPK, mitogen-activated protein kinase; PAGE, polyacrylamide gel electrophoresis; ECL, enhanced chemiluminescence; PKAc, protein kinase A catalytic subunit. 0022-3565/99/2913-1156$03.00/0 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 291, No. 3 Copyright © 1999 by The American Society for Pharmacology and Experimental Therapeutics Printed in U.S.A. JPET 291:1156–1163, 1999 1156 at A PE T Jornals on A uust 4, 2017 jpet.asjournals.org D ow nladed from is an autocrine/paracrine factor secreted by activated T cells (AT). IL-2 gene expression is tightly regulated, and the minimal essential promoter region possesses binding sites for several inducible transcription factors, including AP-1, nuclear factor (NF)-kB, and nuclear factor of activated T cells (NF-AT). Although the IL-2 promoter lacks a consensus CRE site, recent reports have demonstrated a positive role for CREB in T cell activation and IL-2 expression. For example, thymocytes expressing a dominant negative form of CREB exhibited a marked inhibition of IL-2 secretion and proliferation (Barton et al., 1996). CREB/ATF proteins also have been identified in activator protein-1 (AP-1)p and CD28response element (CD28RE) binding complexes within the IL-2 promoter of AT, further supporting the involvement of CREB in IL-2 regulation (Chen and Rothenberg, 1993; Butscher et al., 1998). Phosphorylation of CREB also has been detected after activation of T cells by a variety of stimuli, including phorbol ester (phorbol-12-myristate-13-acetate, PMA) plus calcium ionophore (PMA/Io) and anti-CD3 plus anti-CD28 (Barton et al., 1996; Hsueh et al., 1997). Furthermore, a transient increase in intracellular cAMP levels has been observed immediately after lymphocyte activation with mitogens or phorbol ester/calcium ionophore (Russell, 1978; Kaminski et al., 1994) suggesting a positive role for the cAMP pathway during T cell activation. The NF-kB/c-Rel family of transcription factors also is involved in IL-2 regulation by binding to the kB and CD28RE motifs within the IL-2 promoter (Ghosh et al., 1993). This family of transcription factors is composed of several proteins, including p50, p65, c-Rel, and RelB that can form homoor heterodimers with one another. These dimers are anchored in the cytosol of quiescent cells by IkB inhibitor proteins, and IkB-a is the best characterized of these regulatory proteins (May and Ghosh, 1998). Upon cellular activation, IkB-a is phosphorylated on Ser32 and Ser36 leading to ubiquitination and degradation by the 26S proteosome, enabling NF-kB translocation into the nucleus where it binds to kB motifs in DNA (Brown et al., 1995). NF-kB can be induced by a variety of stimuli, including cytokines, lipopolysaccharide, reactive oxygen species, cAMP elevating agents, and PMA/Io (Shirakawa and Mizel, 1989; Barnes and Karin, 1997). Recently, a large IkB kinase complex has been identified containing two IkB kinases (IKKa and IKKb) that phosphorylate IkB-a after cellular activation (DiDonato et al., 1997; Regnier et al., 1997; Zandi et al., 1997). Earlier studies with D-tetrahydrocannabinol (THC) have characterized the T cell as a sensitive target to inhibition by cannabinoids (Schatz et al., 1993). The objective of the present studies was to further investigate the mechanism of cannabinoid-mediated modulation of T cell activation. Toward this end, experiments were performed to identify the specific CREB/ATF and NF-kB transcription factors modulated by CBN and to examine the up-stream regulation of these transcription factors in the presence of CBN after PMA/Io activation of thymocytes. Materials and Methods Animals. Virus-free female B6C3F1 mice, 6 weeks of age, were purchased from the National Cancer Institute (Bethesda, MD). On arrival, mice were randomized, transferred to plastic cages containing a sawdust bedding (5 mice/cage), and given food (Purina certified laboratory chow) and water ad libitum. Animal holding rooms were kept at 21–24°C and 40 to 60% relative humidity with a 12-h light/